System and method for liver cell culture and maturation

Abstract

The present invention relates to systems and methods for maturation, proliferation and maintenance of function in cells presenting hepatocyte characteristics and differentiated from stem cells. The cells of the present invention may be generated from stem cell grown in collagen sandwich configuration in the presence of a morphogen (e.g. S-NitrosoAcetylPenicillamine (SNAP) or Oncostatin-M (OSM)).

1 . An isolated cell comprising
 a maturated hepatocytic stem cell exhibiting detoxification characteristics wherein the hepatocytic stem cell is maturated from a differentiated stem cell cultured in the presence of S-NitrosoAcetylPenicillamine or Oncostatin-M.   
     
     
         2 . The isolated cell of  claim 1  wherein the differentiated stem cell is selected from the group consisting of human embryonic stem cells, murine embryonic stem cells, and human umbilical cord cells. 
     
     
         3 . The isolated cell of  claim 1  wherein the differentiated stem cell is an embryoid body-mediated heptaocyte-like stem cell. 
     
     
         4 . The isolated cell of  claim 1  wherein the maturated hepatocytic stem cell exhibits CYP expression so as to facilitate detoxification. 
     
     
         5 . The isolated cell of  claim 1  wherein the differentiated stem cell is cultured in or on a collagen matrix. 
     
     
         6 . The isolated cell of  claim 1  wherein the differentiated stem cell is cultured in a collagen sandwich configuration. 
     
     
         7 . A method of producing a maturated hepatocytic cell comprising providing a differentiated stem cell having heptocyte-like characteristics; and culturing the differentiated stem cell in the presence of S-NitrosoAcetylPenicillamine or Oncostatin-M. 
     
     
         8 . The method of  claim 7  wherein the differentiated stem cell is selected from the group consisting of human embryonic stem cells, murine embryonic stem cells, and human umbilical cord cells. 
     
     
         9 . The method of  claim 7  wherein the differentiated stem cell is an embryoid body-mediated heptocyte-like stem cell. 
     
     
         10 . The method of  claim 7  wherein the maturated hepatocytic stem cell exhibits CYP expression so as to facilitate detoxification. 
     
     
         11 . The method of  claim 7  wherein the differentiated stem cell is cultured in or on a collagen matrix. 
     
     
         12 . The method of  claim 7  wherein the differentiated stem cell is cultured in a collagen sandwich configuration. 
     
     
         14 . An implantable tissue construct comprising maturated hepatocytic stem cells exhibiting hepatocyte-like characteristics and detoxification characteristics wherein the hepatocytic stem cells are maturated from a differentiated stem cell cultured in the presence of S-NitrosoAcetylPenicillamine or Oncostatin-M. 
     
     
         15 . A tissue construct for use in extracorporeal liver assist devices comprising; maturated hepatocytic stem cells exhibiting hepatocyte-like characteristics and detoxification characteristics wherein the hepatocytic stem cells are maturated from a differentiated stem cell cultured in the presence of S-NitrosoAcetylPenicillamine or Oncostatin-M. 
     
     
         16 . A population of cultured cells, derived from embryoid bodies, wherein within said population:
 a) at least about 10% of cells comprising said population are positive for intracellular CK 18 expression after six days in culture; or   b) at least about 10% of cells comprising said population are positive for intracellular CK 18 expression after eight days in culture; or   c) at least about 10% of cells comprising said population are positive for intracellular CK 18 expression after ten days in culture.   
     
     
         17 . The population of  claim 16 , wherein
 a) at least about 15% of cells comprising said population are positive for intracellular CK 18 expression after six days in culture; or   b) at least about 15% of cells comprising said population are positive for intracellular CK 18 expression after eight days in culture; or   c) at least about 15% of cells comprising said population are positive for intracellular CK 18 expression after ten days in culture.   
     
     
         18 . The population of  claim 17 , wherein
 a) at least about 20% of cells comprising said population are positive for intracellular CK 18 expression after six days in culture; or   b) at least about 20% of cells comprising said population are positive for intracellular CK 18 expression after eight days in culture; or   c) at least about 20% of cells comprising said population are positive for intracellular CK 18 expression after ten days in culture.   
     
     
         19 . The population of  claim 16 , wherein no more than about 30% of cells comprising said population are positive for intracellular CK 18 expression. 
     
     
         20 . The population of  claim 18 , wherein at least about 30% of cells comprising said population are positive for intracellular CK 18 expression after ten days in culture. 
     
     
         21 . The population of  claim 20 , wherein at least about 40% of cells comprising said population are positive for intracellular CK 18 expression after ten days in culture. 
     
     
         22 . The population of  claim 20 , wherein no more than about 60% of cells comprising said population are positive for intracellular CK 18 expression. 
     
     
         23 . A population of cultured cells, derived from embryoid bodies, said population characterized by secretion of albumin in an amount of at least about 40 ng per 10 6  cells per day after about ten days in culture. 
     
     
         24 . The population of  claim 23 , characterized by secretion of albumin in an amount of no more than about 70 ng per 10 6  cells per day after about ten days in culture. 
     
     
         25 . A population of cultured cells, derived from embryoid bodies, said population characterized by secretion of urea in an amount of at least about 15 ng per 10 6  cells per day after about ten days in culture. 
     
     
         26 . A population of cultured cells, derived from embryonic stem cells characterized by secretion of urea in an amount of no more than about 15 ng per 10 6  cells per day after about ten days in culture. 
     
     
         27 . A population of cultured cells, derived from embryoid bodies, wherein said population, after being cultured for at least about ten days, has cytochrome P450 activity corresponding to at least about 200 uM/ml resorufin after 30 minutes. 
     
     
         28 . The population of cultured cells according to  claim 16 , produced by cultured in a collagen sandwich configuration and further supplemented with a morphogen. 
     
     
         29 . The population of  claim 28 , wherein morphogen is SNAP or OSM. 
     
     
         30 . A method of producing the population of cells according to  claim 16 , comprising:
 a) providing a population of differentiated embryonic stem cells;   b) culturing said population of differentiated embryonic stem cells in a collagen sandwich configuration supplemented with a morphogen.   
     
     
         31 . The method of  claim 30 , wherein said differentiated embryonic stem cells are present in a form of embryoid bodies. 
     
     
         32 . The method of  claim 31  further comprising a step of forming embryoid bodies from undifferentiated stem cells. 
     
     
         33 . The method of  claim 30 , wherein the morphogen is OSM or SNAP. 
     
     
         34 . The method of  claim 33 , wherein the morphogen is OSM, administered at a concentration of 10 ng/ml. 
     
     
         35 . The method of  claim 33 , wherein the morphogen is SNAP, administered at a concentration of 250 uM. 
     
     
         36 . A tissue construct for use in extracorporeal liver assist devices comprising at least a portion of the population of cells according to  claim 16 . 
     
     
         37 . An implantable tissue construct comprising at least a portion of the population of cells according to  claim 16 .